Intraspecies-Resolution Metabarcoding : automated primer design and a plant pathology case study

Pritchard, Leighton and Humphris, Sonia and Campbell, Emma and Watts, Lauren and Haynes, Ed and Pufal, Hollie and Cahill, Greig and Davey, Triona and Elphinstone, John and Toth, Ian (2021) Intraspecies-Resolution Metabarcoding : automated primer design and a plant pathology case study. In: Microbiology Society Annual Conference 2021, 2021-04-26 - 2021-04-30, Online. (

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We present results demonstrating species-specificity and sub-species resolution by novel, automatically-designed metabarcoding primers for environmental DNA analysis. Conventional metabarcoding remains a cornerstone of rapid, high-throughput environmental DNA (eDNA) community analysis and biodiversity assessment. Standard barcodes such as 16S (prokaryotes) and ITS1 (fungi/oomycetes) have been instrumental in identifying the complex composition of communities using total eDNA. However, standard barcodes have limitations in terms of resolution and quantitation and, though genus-level identification can be reliable, species-level identification is often not possible. To overcome the limitations of resolution, we implemented extensions to the diagnostic primer design tool pdp ( that enable automated design of metabarcoding markers and corresponding primers that are (i) specific to a prescribed taxon at species level and (ii) capable of discriminating between members of the same species. This allows for rapid, high-throughput measurement of diversity below species level for a target organism. We aimed to survey geographical distribution and pathogen transfer of the widepread plant pathogenic bacterium Pectobacterium atrosepticum (Pba). This organism has considerable sub-species taxonomic structure identifiable using MLST and with whole-genome methods, but which is not accessible using standard barcodes. We designed metabarcoding primers (202bp) specific to Pba using `pdp`, and established that these have resolution comparable to eight-gene MLST, revealing sub species-level diversity within single fields, and on the same individual plant host.


Pritchard, Leighton ORCID logoORCID:, Humphris, Sonia, Campbell, Emma, Watts, Lauren, Haynes, Ed, Pufal, Hollie, Cahill, Greig, Davey, Triona, Elphinstone, John and Toth, Ian;